Abstract Background: The X-linked gene FOXP3 is a member of the forkhead-box/winged-helix transcription factor family and responsible for X-linked IPEX (Immunodysregulation, Polyendocrinopathy, and Enteropathy, Xlinked) syndrome in mice and humans. FOXP3 appears to function as the master regulator in the development and function of regulatory T cells. Apart from the intrinsic T-cell function, we have demonstrated that FOXP3 is the first X-linked tumor suppressor for breast cancer. FOXP3 directly targets critical oncogenes HER2/ErbB2 and SKP2 and a tumor suppressor gene p21. More recently, we reported that FOXP3 suppresses prostate cancer by repressing transcription of c-MYC. The potential role in FOXP3-mediated regulation on noncoding RNA has not been studied. Using a microRNA (miRNA) array analysis, we screened all known miRNAs in humans for FOXP3 targets. Remarkably, we observed that FOXP3 drastically induced the expressions of miRNA (miR)-146a/b (7-fold for miR-146a and 14-fold for miR-146b) in human breast cancer cells. This observation was validated by a TaqMan miRNA assay in human breast and prostate cancer cell lines. More importantly, tissue-specific deletion of FoxP3 in mouse prostate tissue caused a significant reduction of miR- 146, the mouse homologue of human miR-146a/b. Accumulating data from others demonstrate that miR- 146a/b inhibit cancer cell proliferation, invasion, and metastasis in human cancers, including breast, prostate, pancreatic cancers and glioma. Moreover, others also showed that miR-146a/b negatively regulate NF-&#954;B activity by inhibiting expression of IRAK1 and TRAF6. However, apart from breast cancer metastasis suppressor 1, transactivators of the miR-146a/b are largely unidentified. Since our preliminary data reveal a strong induction of miR-146a/b by FOXP3 in the cancer cells in vitro and in vivo, it is of great interest to identify the impact of FOXP3-miR146-NF&#954;B axis on tumor suppression. Hypothesis and Goals: The central hypothesis of this project is that the FOXP3-miR146-NF-&#954;B axis plays a critical role in tumor suppressor function of FOXP3. The overall goal of this project is to establish the principle of tumor suppressor relay between FOXP3 and noncoding RNA. This hypothesis will be tested in three specific aims. 1) To identify the functional role of miR-146a/b in FOXP3-mediated tumor suppression. 2) To elucidate the mechanism by which FOXP3 induces miR-146a/b transcriptions. 3) To determine whether the function of miR-146a/b is mediated by repression of NF-&#954;B. Approach: First, we will test the effects of the miR-146a/b on proliferation and survival of breast cancer cells, using miR-146a/b inhibitors. The inhibitors will also used to determine whether the miR-146a/b are required for growth inhibition by FOXP3. Second, we will investigate how FOXP3 induction causes a rapid and progressive induction of miR-146a/b transcripts. Using bioinformatic tools, we have identified the candidate forkheadbinding motifs (RYMAAYA) within potential promoter region of miR-146a (5 sites)/b (3 sites). We will use chromatin immunoprecipitation, electrophoretic mobility shift assay and luciferase reporter assays as well as site-directed mutagenesis to identify the functional forkhead motif. Third, using the miR-146a/b inhibitors, we will determine whether FOXP3-induced miR-146a/b represses the NF-&#954;B activity in cancer cells. Once our in vitro studies provide evidence for the FOXP3-miR146-NF&#954;B axis, we will take advantage of the genetic model to determine the functional role of this axis in vivo. Innovation and Significance: Our proposed studies build on a novel observation. If validated, we would have provided a missing link between the X-linked tumor suppressor gene and noncoding RNA and a novel mechanism for regulating miR-146a/b by FOXP3 in tumor suppression. Since lineage-specific ablation of FoxP3 in the mouse prostate epithelial cells leads to prostate hyperplasia and prostatic intraepithelial neoplasia, this mechanism should have a substantial impact on our understanding of early stage cancers and have a potential significance in improving early cancer detection, intervention, and prevention. Furthermore, defective FOXP3 function may provide a novel mechanism for constitutive NF-&#954;B activity in tumor cells, which is still largely unexplained. Linking tumor suppressor function of FOXP3 to NF-&#954;B activation may provide a new therapeutic approach for cancers with FOXP3 defects. Since an NF-&#954;B inhibitor is already available for cancer therapy in human, our hypothesis, if validated by the proposed work, would suggest that cancer with identifiable FOXP3 defects may be responsive to the inhibitor.